Over 100 years ago Starling deduced that hydrostatic and protein osmotic pressures on either side of the capillary barrier control filtration and absorption. Until recently, the assumption has been that the barrier itself does not participate in fluid balance. With modem scientific methods it has become eminently clear that capillaries sense humoral and metabolic signals, which originate from distant organs or locally from tissues that surround the microcirculation. Consequently, a very different picture of a dynamic capillary barrier is coming to the forefront. The long-term goal of the N's laboratory focuses on mechanical stimulation of capillaries. The mechanical stimuli of interest are fluid shear stress and fluid acceleration, two forces imparted to the capillary wall by flowing blood. Measurements of hydraulic conductivity (Lp) will be performed on capillaries located in a living preparation of frog mesentery and cannulated individually with glass micropipettes. In this model, capillary pressure, surface area, network location, blood flow rate, and direction are known. In SPECIFIC AIM 1 we examine the new and exciting possibility that absolute values of capillary Lp relate directly to very SLOW rates of fluid acceleration through the capillary (30 to 50 ums-2). SPECIFIC AIM 2 focuses on the glycocalyx, a layer of glycoproteins located on the capillary lumenal surface, and its potential role in modulating the response of intact capillaries to fluid acceleration. Prostacyclin is one autocoid known to be released acutely (sec to min) upon stimulating cultured endothelial cells with a change in fluid shear stress. SPECIFIC AIM 3 focuses on cyclooxygenase activity (COX), prostacyclin, and the second messenger, adenosine 3'5'-cyclic monophosphate (cAMP) as essential for protecting the capillary barrier in the face of changes in blood flow. Finally, in SPECIFIC AIM 4, we propose two unique mechanisms for sensing fluid stimuli in vivo. Partial digestion of the glycocalyx will be combined with inhibition of COX. Capillaries will be challenged with SLOW (5 min) or ABRUPT (< 0.1 s) changes in fluid velocity to reveal lumenal versus whole cell mechanisms for sensing flow. Collectively, the results from these studies will be critical to our understanding of capillary barrier function, mechanotransduction in vivo, and whole body fluid balance. Chronic venous insufficiency, congestive heart failure, and lymphedema represent three human diseases where the capillary barrier is not functioning properly and mechanical stimulation of the microcirculation may exacerbate the problem. The work proposed here will impact directly on clinical research efforts focused on these costly and potentially life-threatening diseases.